US20240295567A1 - Hypoxia biomarker and use thereof - Google Patents

Hypoxia biomarker and use thereof Download PDF

Info

Publication number
US20240295567A1
US20240295567A1 US18/574,504 US202218574504A US2024295567A1 US 20240295567 A1 US20240295567 A1 US 20240295567A1 US 202218574504 A US202218574504 A US 202218574504A US 2024295567 A1 US2024295567 A1 US 2024295567A1
Authority
US
United States
Prior art keywords
spink1
hypoxia
pathological condition
exacerbation
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
US18/574,504
Other languages
English (en)
Inventor
Hiroshi Harada
Minoru Kobayashi
Tatsuya Suwa
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kyoto University NUC
Original Assignee
Kyoto University NUC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kyoto University NUC filed Critical Kyoto University NUC
Assigned to KYOTO UNIVERSITY reassignment KYOTO UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HARADA, HIROSHI, KOBAYASHI, MINORU, SUWA, TATSUYA
Publication of US20240295567A1 publication Critical patent/US20240295567A1/en
Pending legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/99—Enzyme inactivation by chemical treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification

Definitions

  • the present invention relates to a hypoxia biomarker and use thereof.
  • hypoxic state in vivo was heretofore known to be associated with various pathological conditions.
  • a hypoxic state an area at a low oxygen partial pressure
  • a hypoxic state is an important environmental factor that induces the resistance of cancer cells to radiotherapy and anticancer drug therapy.
  • the lower the oxygen concentration the worse the pathological condition. Accordingly, in order to comprehend and predict pathological conditions and therapeutic efficacy on the pathological conditions, it is important to know, for example, the extent of hypoxic areas and the degree of hypoxia.
  • NPL Non-patent Literature
  • an immunostaining method comprising sampling a part of tissue from a living body and targeting, for example, CA9 (carbonic anhydrase 9) or GLUT1 (glucose transporter 1) in the sampled tissue.
  • CA9 carbonic anhydrase 9
  • GLUT1 glucose transporter 1
  • An object of the present disclosure is to provide a novel marker useful for measuring hypoxia and predicting the risk of exacerbation of a hypoxia-related pathological condition; a method for measuring hypoxia using the marker as an indicator; a method for predicting the risk of exacerbation of a pathological condition using the marker as an indicator; a composition for measuring hypoxia or predicting the risk of exacerbation of a pathological condition; a method for screening for a therapeutic agent for a hypoxia-related pathological condition; a composition for ameliorating a hypoxia-related pathological condition; and the like.
  • the present inventors conducted extensive research and found that the expression level and the secretion level of SPINK1 (serine protease inhibitor Kazal type I) increase in a hypoxia stimulus-dependent manner.
  • the present inventors further found that SPINK1 is involved in the exacerbation of a pathological condition.
  • the present inventors further found that SPINK1 is useful as a biomarker for hypoxia in a subject or as a biomarker for the exacerbation of a pathological condition.
  • the present invention has been accomplished as a result of further research based on the above findings.
  • the present invention includes, for example, the following embodiments.
  • Item 1 A method for measuring hypoxia in a subject, comprising measuring the amount of SPINK1 (serine protease inhibitor Kazal type I) in a sample collected from the subject.
  • SPINK1 serine protease inhibitor Kazal type I
  • Item 2 A method for predicting the risk of exacerbation of a pathological condition, comprising measuring the amount of SPINK1 in a sample collected from a subject.
  • Item 3 The method according to Item 1 or 2, wherein the sample is at least one member selected from the group consisting of blood, plasma, serum, urine, milk, saliva, cell specimens, and tissue specimens.
  • Item 4 Use of SPINK1 as a biomarker for measuring hypoxia or predicting the risk of exacerbation of a pathological condition.
  • Item 5 A composition for measuring hypoxia or predicting the risk of exacerbation of a pathological condition, the composition comprising a SPINK1 detection reagent.
  • Item 6 A method for screening a candidate substance for a therapeutic agent for a hypoxia-related pathological condition, the screening being performed by using, as an indicator, whether or not the candidate substance can inhibit SPINK1.
  • Item 7 A composition for ameliorating a hypoxia-related pathological condition, the composition comprising a SPINK1 inhibitor.
  • Item 8 The method according to Item 2 or 3, the use according to Item 4, the composition according to Item 5, the method according to Item 6, or the composition according to Item 7, wherein the pathological condition is at least one member selected from the group consisting of tumors and ischemic diseases.
  • the present invention can provide a method of measuring hypoxia and a method for predicting the risk of exacerbation of a pathological condition by using SPINK1 as an indicator.
  • the present invention can provide a hypoxia biomarker useful for measuring hypoxia or predicting the risk of exacerbation of a pathological condition.
  • the present invention can provide a composition for measuring hypoxia or predicting the risk of exacerbation of a pathological condition, the composition comprising a SPINK1 detection reagent; a method for screening a candidate substance for a therapeutic agent for a hypoxia-related pathological condition, the screening being performed by using, as an indicator, whether or not the candidate substance can inhibit SPINK1; a composition for ameliorating a hypoxia-related pathological condition, the composition comprising a SPINK1 inhibitor; and the like.
  • FIG. 1 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 2 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 3 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 4 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 5 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 6 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 7 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 8 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 9 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 10 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 11 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 12 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 13 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 14 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 15 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 16 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 17 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 18 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 19 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 20 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 21 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 22 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 23 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 24 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 25 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 26 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 27 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 28 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 29 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 30 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 31 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 32 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 33 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 34 is a diagram showing examples of SPINK1 reported in the NCBI.
  • FIG. 35 is a diagram showing one example of SPINK1 reported in the NCBI.
  • FIG. 36 is a diagram showing the results of SPINK1 mRNA expression in a hypoxia stimulus-dependent manner.
  • FIG. 37 is a diagram showing the results of SPINK1 mRNA expression and SPINK1 protein secretion in a hypoxia stimulus-dependent manner.
  • FIG. 38 is a diagram showing that SPINK1 reflects the amount of hypoxia in tumors.
  • FIG. 39 is a diagram showing that SPINK1 is secreted from cells in a hypoxic state and is present in or around the cells.
  • FIG. 40 is a diagram showing that an increase of SPINK1 contributes to the exacerbation of a pathological condition.
  • FIG. 41 is a diagram showing that SPINK1 can be used as an indicator of the exacerbation of a hypoxia-related pathological condition.
  • FIG. 42 is a diagram showing that an increase of SPINK1 contributes to exacerbation of a pathological condition.
  • FIG. 43 is a diagram showing that inhibition of SPINK1 can inhibit the exacerbation of a pathological condition.
  • FIG. 44 is a diagram showing that a SPINK1 high-value group has a lower overall survival rate than a SPINK1 low-value group.
  • the method for measuring hypoxia in a subject comprises the step of measuring the amount of SPINK1 (serine protease inhibitor Kazal type I) in a sample collected from a subject.
  • SPINK1 serine protease inhibitor Kazal type I
  • SPINK1 is known as a serine protease inhibitor Kazal type I.
  • SPINK1 can be known, for example, from known databases, such as those shown in FIGS. 1 to 35 .
  • FIGS. 1 to 35 are examples of reports on SPINK1 of humans or other species shown in the NCBI.
  • the report on SPINK1 Homo sapiens (human) ID: 6690), which is described first among the reports shown in FIGS. 1 to 35 , is information updated in May 2021.
  • the others are examples of SPINK1 of rats or other species reported at the same point in time, i.e., in May 2021.
  • SPINK1 can be any SPINK1 and is not limited to the specific SPINK1 shown in these figures.
  • the sample collected from a subject is not limited as long as it is a target of measurement of hypoxia.
  • samples include blood, plasma, serum, saliva, milk, urine, stool, tear, sweat, hair, body hair, spinal fluid, bone marrow fluid, joint fluid, sputum, nasal discharge, sebum, lymph fluid, tissues and cells of pathological sites (e.g., tissue specimens and cell specimens, such as tumor tissues, tumor cells, and myocardial cells); preferably biological samples such as blood, plasma, serum, urine, milk, saliva, tissue specimens, and cell specimens; and more preferably biological samples, such as blood, plasma, serum, saliva, milk, and urine from the viewpoint of being less invasive to subjects.
  • the pathological site such as tumor tissue, can be, for example, benign or malignant. Collecting a sample from a subject can be performed in accordance with known procedures. Samples can be used alone or in a combination of two or more.
  • subjects include mammals such as humans, monkeys, mice, rats, rabbits, goats, sheep, horses, guinea pigs, hamsters, dogs, cats, monkeys, and elephants; preferably humans, monkeys, mice, and rats; and more preferably humans.
  • the procedures for measuring the amount of SPINK1 in the sample are not limited.
  • the measurement can be performed according to known procedures, and the amount of SPINK1 can be measured directly or indirectly.
  • examples of SPINK1 measured include mRNA precursors, mRNA, proteins, and activity.
  • any object that changes in accordance with changes in the amount of SPINK1 may be used as the target of measurement, and the amount of SPINK1 may be indirectly measured based on, for example, the measurement results of the object.
  • the procedures for the measurement are not limited.
  • Examples include known gene detection methods and immunological methods; for example, for the measurement of mRNA precursors and mRNAs, examples include in situ hybridization, RT (Reverse Transcription)-PCR (including quantitative RT-PCR), microarray, RNA-Seq, and Northern blotting; and for the measurement of proteins, examples include immunostaining, Western blotting, ELISA (enzyme-linked immuno-sorbent assay), flow cytometry, dot blotting, and mass spectrometry.
  • the procedures for the measurement can be appropriately determined according to the target of measurement. Samples may be directly subjected to SPINK1 measurement as is, or may be pretreated as necessary.
  • examples of primers for quantitative RT-PCR include SPINK1 Human qPCR Primer Pair (NM_003122) (Catalog #24833) produced by OriGene Technologies, Inc.
  • examples of antibodies include Anti-SPINK1/P12 antibody (catalog #ab183034) produced by Abcam PLC.
  • examples of ELISA kits include Human SPINK1 DuoSet ELISA (catalog #DY7496-05) produced by R & D Systems.
  • the measurement of the amount of SPINK1 further means detection. That is, the measurement of the amount of SPINK1 may be to comprehend whether SPINK1 is present or absent in a sample, or may be to comprehend the amount of SPINK1 in a sample. In the present disclosure, the amount measured may be an absolute amount or a relative amount.
  • the measurement method of the present disclosure may further comprise the step of setting a reference value for the amount of SPINK1 and comparing the reference value with the amount of SPINK1 (measured value) in a sample actually measured in the manner as described above.
  • the reference value may be arbitrarily set according to the purpose.
  • the reference value may be the amount of SPINK1 in a sample collected from a person whose oxygen concentration is a normal value (e.g., a sample derived from a healthy person, a sample derived from a person who has no pathological condition that causes a decrease in oxygen concentration).
  • the amount of SPINK1 (measured value) in a sample collected from a subject is higher than the reference value, the subject from whom the sample was collected is determined to have a site with an oxygen concentration lower than normal oxygen concentration; and if the measured value is as low as or lower than the reference value, the subject from whom the sample was collected is determined to be normal in terms of oxygen concentration.
  • the reference value may be the amount of SPINK1 in a sample collected from a person whose oxygen concentration is lower than a normal value (e.g., a sample derived from a person with a pathological condition).
  • a normal value e.g., a sample derived from a person with a pathological condition.
  • the subject from whom the sample was collected is determined to have a site with an oxygen concentration as low as or lower than that of the provider of the reference value (e.g., a person with a pathological condition), or have more sites with such an oxygen concentration than the provider of the reference value.
  • the subject from whom the sample was collected can be determined to have no site with an oxygen concentration lower than that of the provider of the reference value or have fewer sites with an oxygen concentration as low as or lower than that of the provider of the reference value.
  • such a reference can be used to determine the degree of hypoxia in a subject.
  • the measured value and the reference value are not limited.
  • the measurement is preferably made by using the same type of sample (for example, if the sample used to obtain a measured value is blood, the sample used to obtain the reference value is also blood).
  • the measurement of the amount of SPINK1 is also performed by using the same measurement procedure.
  • the reference value may also be a value obtained by pre-measuring a sample collected from a subject.
  • SPINK1 if SPINK1 is detected in a sample, it can be determined that the subject from whom the sample was collected has a high probability of having a site in a hypoxic state (an area with a reduced oxygen partial pressure). If SPINK1 is not detected, it is determined that the subject from whom the sample was collected has no site in a hypoxic state.
  • the subject from whom the sample was collected is determined to have a site with a lower concentration, or have more sites in a hypoxic state; and when a sample has a smaller amount of SPINK1, the subject from whom the sample was collected is determined to have an oxygen concentration closer to the normal oxygen concentration, or have fewer sites in a hypoxic state.
  • the measurement method of the present disclosure may optionally further comprise such a determination step etc.
  • the relationship between the degree of hypoxia (oxygen concentration) and the severity of a pathological condition (exacerbation) may vary depending on the type of pathological condition (disease) and other factors. Therefore, the reference value may be appropriately determined depending on the type of pathological condition (disease) and other factors.
  • the reference value may be an absolute value or a relative value.
  • the measurement method of the present disclosure it is possible to know the presence or absence of SPINK1 and/or the amount of SPINK1 in a sample. Therefore, the measurement method is useful in that the presence or absence of SPINK1 and/or the amount of SPINK1 in a sample can be comprehended.
  • the present inventors found that there is a correlation between hypoxia and the amount of SPINK1. The present inventors further found that the lower the oxygen concentration, the larger the amount of SPINK1 expressed, secreted, etc.
  • the amount of SPINK1 in the living body tends to increase in a hypoxia concentration-dependent manner (the lower the oxygen concentration and/or the more hypoxia sites (areas) in the living body, the larger the increase in the amount of SPINK1). Further, it is said that the lower the oxygen concentration, the worse or the more refractory the pathological condition (the higher the risk of exacerbation). It can be said from this that the degree of hypoxia in a subject can be known by measuring the amount of SPINK1 in a sample collected from a subject.
  • areas at a reduced oxygen partial pressure in a subject e.g., the presence or absence of such hypoxic areas and the degree of hypoxia
  • the present disclosure may also be said to provide a method for measuring hypoxia in a subject based on the amount of SPINK1 in a sample.
  • the measurement method of the present disclosure is also useful in that one can know, for example, the degree of hypoxia in a subject, based on the amount of SPINK1 in a sample. Further, as described below, the method is also useful in that an exacerbated pathological condition or the risk of exacerbation can be predicted by using the amount of SPINK1 as an indicator. This prediction is also useful in investigating and performing a treatment for a pathological condition according to the subject. The pathological condition is explained as described below.
  • the present disclosure further includes a method for predicting the risk of exacerbation of a pathological condition, comprising the step of measuring the amount of SPINK1 in a sample collected from a subject.
  • the subject, sample, SPINK1, measurement, and the like are all explained as described above in the measurement method. It is conventionally said that the lower the oxygen concentration, the worse the pathological condition.
  • the prediction method of the present invention it can be predicted from this that the larger the amount of SPINK1 in a sample, the higher the risk of exacerbation of a pathological condition in the subject; and the smaller the amount of SPINK1 in a sample, the lower the risk of exacerbation of a pathological condition in the subject.
  • preferable examples of the amount of SPINK1 to be measured in the prediction method include the mRNA level and the protein level, and more preferably the protein level.
  • pathological conditions include tumors and ischemic diseases such as ischemic heart diseases (e.g., angina pectoris and myocardial infarction) and ischemic brain diseases (e.g., cerebral infarction); and preferably tumors, myocardial infarction, and cerebral infarction.
  • tumors include brain cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, lung cancer, stomach cancer, colon cancer, liver cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, bone tumor, and skin cancer.
  • pathological conditions pathological sites, not in a healthy condition
  • pathological conditions are usually considered to be in a hypoxic state.
  • the risk of exacerbation means the degree of progression or refractory level of a pathological condition; and further means malignancy (malignancy level), for example, in the case of tumors. It can be said that the higher the risk of exacerbation, the further the pathological condition is progressing, and the more refractory (harder to cure) the pathological condition.
  • examples of the exacerbation of a pathological condition include radiation resistance of tumor tissue, which is refractory.
  • hypoxia is not limited as long as it is within the range of hypoxia known in this technical field.
  • hypoxia can be explained as an oxygen concentration being below the normal oxygen partial pressure in each organ, and the oxygen concentration is preferably, for example, in the range of 5% or less, 3% or less, 1% or less, or 0.1% or less.
  • the lower limit of the oxygen concentration is theoretically 0%.
  • the oxygen concentration is a value obtained by measurement procedures usually used in this technical field. This explanation is also applicable to the measurement method described above.
  • the risk of exacerbation of a pathological condition can be predicted based on the amount of SPINK1 measured.
  • the larger the amount of SPINK1 in a sample the higher the risk of exacerbation of a pathological condition in a subject from whom the sample was collected (there is a high probability that the pathological condition is progressing, is highly malignant, or is refractory) (that is, a high risk of exacerbation of a pathological condition is predicted); and it is determined that the smaller the amount of SPINK1, the lower the risk of exacerbation of a pathological condition in a subject from whom the sample was collected (that is, a low risk of exacerbation of a pathological condition is predicted).
  • the prediction method of the present disclosure may further comprise the step of setting a reference value and comparing the reference value with the amount of SPINK1 in a sample measured in the manner as described above (the measured value).
  • the reference value may be arbitrarily set according to the purpose. For example, in a case in which the amount of SPINK1 in a sample that has been already determined to have a low risk of exacerbation is used as a reference value, if the amount of SPINK1 (the measured value) in a sample collected from a subject is higher than the reference value, then the subject from whom the sample was collected is determined to have a high probability of having exacerbation of a pathological condition; and if the measured value is as low as or lower than the reference value, then the subject from whom the sample was collected is determined to have a low probability of having exacerbation of a pathological condition.
  • the amount of SPINK1 in a sample that has been already determined to have a high risk of exacerbation is used as a reference value
  • the amount of SPINK1 (the measured value) in a sample collected from a subject is as high as or higher than the reference value
  • the subject from whom the sample was collected is determined to have a high probability of having exacerbation of a pathological condition
  • the measured value is smaller than the reference value, then the subject from whom the sample was collected is determined to have a low probability of having exacerbation of a pathological condition.
  • the measured value and the reference value are not limited.
  • the measured value and the reference value are values obtained by using the same type of sample (for example, if the sample used to obtain the measured value is blood, then the sample used to obtain the reference value is also blood), and the amount of SPINK1 is measured in accordance with the same measurement procedures.
  • the reference value may also be a value obtained by pre-measuring a sample collected from a subject.
  • if SPINK1 is detected in a sample it can be determined that a subject from whom the sample was collected has a high probability of having a site in a hypoxic state; and if SPINK1 is not detected in a sample, a subject from whom the sample was collected is determined to have no site in a hypoxic state.
  • a high risk of exacerbation of a pathological condition is predicted; in the latter case, a low risk of exacerbation or no pathological condition (pathological site) is predicted.
  • the prediction method of the present disclosure may optionally further comprise, for example, the step of making such a determination.
  • the relationship between the degree of hypoxia (oxygen concentration) and the severity of a pathological condition (exacerbation) may vary depending on the type of pathological condition (disease) and other factors. Therefore, the reference value may be appropriately determined depending on, for example, the condition of a subject from whom a sample is collected, the type of pathological condition (disease), and the severity of the pathological condition. Thus, the reference value may be appropriately determined by considering the amount of SPINK1 in a sample derived from, for example, a healthy person or a patient already determined to have a pathological condition.
  • the reference value may be determined by a method different from that of the present disclosure or may be determined beforehand by the method of the present disclosure, or may be based on a value shown in databases regarding pathological conditions, oxygen concentration and/or the amount of SPINK1.
  • the reference value may be a value derived from a subject who is subjected to prediction, or may be a value derived from a third party who is different from the subject.
  • the reference value may also be an absolute value or a relative value. This explanation also applies to the measurement method described above.
  • a subject from whom a sample is collected may be a subject having some pathological condition (disease), may be a subject suspected of having a pathological condition (disease), may be a subject whose pathological condition (disease) is unknown, or may be a subject with no pathological condition (disease).
  • the lower the oxygen concentration the worse the pathological condition.
  • the oxygen concentration is low at or around a tumor site.
  • the lower the oxygen concentration the more resistant the tumor is to radiation therapy.
  • a low oxygen concentration is known to promote cancer invasion etc.
  • Myocardial infarction is mainly caused by lowering of the oxygen concentration due to obstruction of blood flow etc.
  • various pathological conditions are known to closely relate to the oxygen concentration at or around the pathological site.
  • it is also necessary to select an appropriate therapeutic method according to the degree of hypoxia as is typically shown by the fact that radiation resistance is observed in tumors having a reduced oxygen concentration.
  • the prediction method of the present disclosure it is possible to predict the risk of exacerbation of various pathological conditions based on the presence or absence of SPINK1 in a sample and/or the amount of SPINK1 in a sample.
  • the prediction method of the present disclosure is useful in this point.
  • the prediction of the risk of exacerbation of a pathological condition is useful in considering whether or not a treatment is necessary for the subject and what therapeutic method is to be selected.
  • SPINK1 is useful as a biomarker for measuring hypoxia or for predicting the risk of exacerbation of a pathological condition.
  • SPINK1 can also be referred to as a hypoxia marker.
  • SPINK1 is explained as described above.
  • preferable examples of biomarkers include SPINK1 mRNA and SPINK1 protein.
  • blood or the like can also be used as measurement samples. Therefore, this marker can also be used, for example, as a blood marker.
  • the determination of a therapeutic effect includes a determination of whether or not the treatment is efficacious. For example, if an increase in the amount of SPINK1 in a sample is suppressed or the amount of SPINK1 decreases, the treatment is determined to be efficacious. If an increase in the amount of SPINK1 is not suppressed or the amount of SPINK1 increases, the treatment is determined to be inefficacious.
  • the prognosis prediction is a determination of whether the prognosis is good or not.
  • the prognosis is predicted to be good.
  • the prognosis is predicted to be bad.
  • the present disclosure further includes a composition for measuring hypoxia or for predicting the risk of exacerbation of a pathological condition, the composition comprising a SPINK1 detection reagent.
  • the SPINK1 The SPINK1, the measurement of hypoxia, and the prediction of the risk of exacerbation of a pathological condition are as described above.
  • the SPINK1 detection reagent is not limited as long as it can detect SPINK1.
  • examples include primers, probes, antibodies, aptamers (e.g., nucleic acid aptamers), compounds (e.g., natural compounds and synthetic compounds), and the like capable of specifically detecting SPINK1.
  • Usable antibodies can be any antibody, such as polyclonal antibodies, monoclonal antibodies, and fragments thereof (e.g., Fab, Fab′, and F (ab′) 2 ).
  • the detection reagent to be used may any reagent that can directly or indirectly detect the mRNA precursor, mRNA, protein, activity, etc., of SPINK1, as described above, and is not limited as long as the presence etc. of SPINK1 can be comprehended.
  • reagents may also be labeled with, for example, an enzyme or a fluorescent dye, may bind to other proteins, etc., or may be immobilized on microplates, magnetic beads, or the like as needed.
  • the detection reagent may be a conventionally known SPINK1 detection reagent.
  • examples include the primers, antibodies, and ELISA kits described above.
  • the composition may further contain optional components as long as such components do not interfere with the effect of the present disclosure.
  • optional components include carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickening agents, colorants, fragrances, chelating agents, pH adjusters, and preservatives.
  • Such optional components may be used alone or in a combination of two or more.
  • the amount of optional components added can be appropriately determined within the range that they do not interfere with the effects of the present disclosure.
  • the content of the SPINK1 detection reagent in the composition is not limited as long as SPINK1 can be detected.
  • the content can be appropriately determined according to, for example, the type of primer, probe, antibody, or the like used.
  • the composition may be in the form of a solid (e.g., powders, granules, and tablets), a semi-solid, or a liquid.
  • the detection using the composition can be performed with reference to known detection procedures according to, for example, the types of primers, probe, antibody, activity, target, etc. as described above.
  • the detection can be performed by the same procedures as in the measurement method described above.
  • the composition may be in the form of a kit.
  • the kit may further contain, in addition to the composition, a buffer, a nucleic acid amplification reagent, reverse transcriptase, a substrate, a primary antibody, a secondary antibody, an instruction manual, an aptamer (e.g., nucleic acid aptamer), a compound (e.g., natural compounds and synthetic compounds), and the like usable for detection.
  • the instructions for use may describe the URL of a web page, reading code, or the like, or may be arranged such that the procedures for use, etc. can be obtained via a URL, reading code, etc.
  • the present disclosure can also be said to provide a kit for measuring hypoxia or predicting the risk of exacerbation of a pathological condition.
  • composition and the kit are useful in that the measurement and prediction described above can be easily performed.
  • the present disclosure includes a method for screening a candidate substance for a therapeutic agent for a hypoxia-related pathological condition, the screening being performed using, as an indicator, whether or not the candidate substance can inhibit SPINK1.
  • SPINK1 can be used as an indicator in hypoxia-related pathological conditions, and further found that the larger the amount of SPINK1, the higher the risk of exacerbation of a pathological condition. Further, the present inventors found that inhibiting SPINK1 can mitigate the risk of exacerbation of a pathological condition (e.g., refractory degree), as shown in the Examples below. Thus, drugs capable of inhibiting SPINK1 are useful for improving hypoxia-related pathological conditions and suppressing the progression of hypoxia-related pathological conditions. Thus, screening for therapeutic agents for hypoxia-related pathological conditions can be performed based on whether or not a candidate substance can inhibit SPINK1.
  • a pathological condition e.g., refractory degree
  • Candidate substances are not particularly limited and can be any substance such as compounds (e.g., natural compounds, synthetic compounds, etc.), antibodies, and aptamers (nucleic acid aptamers).
  • Candidate substances can be selected from known substances or can be newly synthesized, produced, etc.
  • the inhibition of SPINK1 includes, for example, being capable of reducing the expression level of SPINK1 mRNA precursor or SPINK1 mRNA, being capable of reducing the expression level of SPINK1 protein, being capable of reducing the secretion level of SPINK1 protein, and being capable of reducing the activity of SPINK1 protein. Further, the inhibition of SPINK1 can also be determined by using, as an indicator, for example, whether or not the activity of SPINK1 (the activity of inducing radiation resistance of cells, the activity of inducing antioxidant capacity of cells, the activity of inducing epidermal growth factor receptor (EGFR) signaling) can be inhibited. Thus, the inhibition of SPINK1 includes inhibition of expression and secretion, and inhibition of activity.
  • EGFR epidermal growth factor receptor
  • hypoxia-related pathological condition is not limited as long as it is a condition in which hypoxia is a causative factor. Examples includes pathological conditions described above.
  • the candidate substance is determined to have a high probability of being useful in treating a hypoxia-related pathological condition; and if a candidate substance cannot inhibit SPINK1, then the candidate substance is determined to have a high probability of not being useful in treating a hypoxia-related pathological condition.
  • the screening method may further comprise this determination step.
  • Whether a candidate substance can inhibit SPINK1 or not can be appropriately determined with reference to known screening procedures according to the candidate substance.
  • the screening method may comprise setting a control value for inhibition and comparing the inhibition achieved by using a candidate substance (a test value) with the control value. For example, when the SPINK1 inhibition level achieved by bringing a substance that does not have a SPINK1-inhibiting effect into contact with a sample is used as a control value, the SPINK1 inhibition level achieved by bringing a candidate substance into contact with a sample (a test value) is compared with the control value. If the test value is higher than the control value (i.e., if a large amount of SPINK1 is inhibited), the candidate substance is determined to have a high probability of inhibiting SPINK1.
  • the candidate substance is determined to have a high probability of being useful in treating a hypoxia-related pathological condition.
  • the test value is as low as or lower than the control value
  • the candidate substance is determined to have a low probability of inhibiting SPINK1.
  • the candidate substance is determined to have a high probability of not being useful in treating a hypoxia-related pathological condition.
  • the SPINK1 inhibition level may be an absolute value or a relative value, and is not limited as long as the presence or absence of inhibition of SPINK1 and/or the degree of inhibition of SPINK1 can be known.
  • the sample is not limited as long as the inhibition can be determined. For example, samples collected from the subjects described above may be used.
  • the value achieved by using a substance that is already known to be useful in the treatment of a hypoxia-related pathological condition or a substance that is already known to have a SPINK1-inhibiting effect may be used as a control value.
  • the SPINK1 inhibition level achieved by bringing such a substance into contact with a sample is used as a control value
  • the SPINK1 inhibition level achieved by bringing a candidate substance into contact with a sample (a test value) is compared with the control value. If the test value is as high as or higher than the control value, the candidate substance is determined to have a high probability of inhibiting SPINK1.
  • the candidate substance is determined to have a high probability of being useful in treating hypoxia-related pathological conditions. If the test value is lower than the control value, the candidate substance is determined to have a low probability of inhibiting SPINK1. Based on this result, the candidate substance may be determined to have a high probability of not being useful in treating a hypoxia-related pathological condition. In this case as well, the inhibition level (inhibition amount) and the sample are explained as described above.
  • the screening method is useful, for example, in searching, creating, or determining a therapeutic agent for a hypoxia-related pathological condition.
  • the present disclosure further includes a composition for ameliorating a hypoxia-related pathological condition, the composition comprising a SPINK1 inhibitor.
  • the present inventors found that inhibiting SPINK1 can mitigate the refractory degree of a hypoxia-related condition.
  • the present disclosure also provides a composition for ameliorating a hypoxia-related pathological condition, the composition comprising a SPINK1 inhibitor.
  • the SPINK1 inhibitor is not limited as long as it can inhibit SPINK1.
  • examples include compounds (e.g., natural compounds and synthetic compounds), antibodies, and aptamers (nucleic acid aptamers); preferably antibodies; and more preferably SPINK1 neutralizing antibodies.
  • the inhibitor may be one capable of inhibiting SPINK1 as stated above. The inhibition of SPINK1 can be explained in the same manner as described above.
  • the content of the SPINK1 inhibitor in the composition is not limited and can be appropriately determined according to, for example, the type of compound, antibody, aptamer, or the like used and inhibition capability.
  • the composition may be in the form of a solid (e.g., powders, granules, or tablets), a semi-solid, or a liquid.
  • the route of administration is also not limited and may be oral or parenteral. Examples of parenteral administrations include subcutaneous, intramuscular, intravenous, sublingual, oral mucosal, transrectal, transvaginal, intranasal, inhalation, spray, and dermal administrations.
  • composition may further optionally contain pharmacologically acceptable components as long as such components do not interfere with the effect of the present disclosure.
  • pharmacologically acceptable components include solvents, pH adjusters, excipients, stabilizers, binders, disintegrants, lubricants, colorants, fragrances, and preservatives.
  • Such components may be used alone or in a combination of two or more.
  • the amounts of components optionally incorporated may also be appropriately selected within the range that they do not interfere with the effect of the present invention.
  • composition for amelioration is useful for ameliorating a hypoxia-related pathological condition.
  • the hypoxia-related pathological condition can be explained as described above in the pathological condition.
  • Human cervical cancer-derived cell line HeLa was sown into a cell culture dish (1 ⁇ 10 5 per well of the 6-well cell culture plate) and cultured overnight in a CO 2 incubator at a normal oxygen concentration (20%). After the cells were then cultured in a CO 2 incubator set to an oxygen concentration shown in FIG. 36 for the periods of time shown in FIG. 36 , the total RNA was collected using Sepasol RNA I Super G (produced by Nacalai Tesque, Inc.) A reverse transcription reaction and quantitative PCR experiments for SPINK1 and CA9 were performed. A quantitative PCR experiment using ACTB as an internal standard was also performed at the same time.
  • FIG. 36 shows the results.
  • FIG. 36 ( a ) shows the expression level of SPINK1 mRNA after 48 hours of exposure of HeLa cells to each oxygen concentration
  • FIG. 36 ( b ) shows the expression level of CA9 mRNA after 48 hours of exposure of HeLa cells to each oxygen concentration
  • FIG. 36 ( c ) shows the expression level of SPINK1 mRNA after 12, 24, and 48 hours of exposure of Hela cells to an oxygen concentration of 20% or 0.1%.
  • the expression level of SPINK1 mRNA and the expression level of CA9 mRNA significantly increased under exposure to an oxygen concentration of 0.1%.
  • the expression level of CA9 which is conventionally known as a hypoxia marker, tended to increase gradually as the oxygen concentration decreased to 3%, 1% and 0.1%, whereas the expression level of SPINK1 at an oxygen concentration of 3% or 1% was as low as it was at an oxygen concentration of 20%, with a sharp increase in the expression level of SPINK1 at an oxygen concentration of 0.1%.
  • Human cervical cancer-derived cell line HeLa, human prostate cancer-derived cell line DU 145 , and human osteosarcoma-derived cell line U2OS were sown into cell culture dishes (1 ⁇ 10 5 cells per well of a 6-well cell culture plate for quantitative RT-PCR and 1.5 to 2.0 ⁇ 10 5 cells per well of a 6-well cell culture plate for ELISA assay), and cultured overnight in a CO 2 incubator at a normal oxygen concentration. Thereafter, after the cell lines were cultured for 48 hours in a CO 2 incubator set to the oxygen concentrations shown in FIG. 37 , the cell culture fluid was collected and ELISA assay for SPINK1 was performed.
  • a quantitative PCR experiment using ACTB as an internal standard was also performed at the same time.
  • FIG. 37 shows the results.
  • FIG. 37 shows the amount of SPINK1 mRNA in the cells (top row) and the amount of SPINK1 protein in the cell culture fluid (bottom row).
  • the SPINK1 mRNA expression level and the SPINK1 protein secretion level significantly increased at an oxygen concentration of 0.1% as compared with those at an oxygen concentration of 20%.
  • Tumor-bearing mice were prepared by subcutaneously transplanting a human cervical cancer-derived cell line HeLa into the right lower-leg thigh. Using the tumor-bearing mice as subjects, the blood vessel in the right lower-leg thigh was ligated to reduce the blood flow to the HeLa-transplanted tumor. After the lapse of time shown in FIG. 38 , the transplanted tumor was removed. The total RNA was collected from the removed transplanted tumor by using Sepasol RNA I Super G (produced by Nacalai Tesque, Inc.). A reverse transcription reaction and a quantitative PCR experiment for SPINK1 were performed. A quantitative PCR experiment using ACTB as an internal standard was also performed at the same time. The total protein was also extracted from the removed transplanted tumor, and ELISA assay for SPINK1 was performed at the same time.
  • FIG. 38 shows the results. As can be understood from FIG. 38 , induction of the expression of SPINK1 mRNA and SPINK1 protein was observed in the biogenic tumor tissue exposed to a hypoxic environment by ligation. As the time of exposure to the hypoxic environment increased, both of the SPINK1 mRNA level and the SPINK1 protein level increased. This confirmed that the amount of SPINK1 increased in a hypoxia exposure-dependent manner.
  • Pimonidazole was administered to tumor-bearing mice that had been prepared by transplanting human cervical cancer-derived cell line HeLa. Sixty minutes after the administration, the transplanted tumor was removed. This tumor was fixed in a 10% neutral buffered formalin solution and then paraffin-embedded to prepare tumor sections. The paraffin sections were used in an immunohistochemical staining experiment using an anti-pimonidazole antibody (mouse monoclonal antibody; Hypoxyprobe, Inc., Catalog #HP2-1000) and anti-SPINK1 antibody (rabbit monoclonal antibody; EPR12696 (2), Abcam, Catalog #ab183034). After staining with each antibody, nuclei were stained using Hoechst 33342.
  • FIG. 39 shows the results.
  • stained portions in the upper photo “Pimonidazole” indicate hypoxic areas; stained portions in the photo “SPINK1” indicate that SPINK1 protein is present; and stained portions in the photo “Hoechst” indicate that tumor cells are present.
  • the lower photo in FIG. 39 is a superimposition of these photos.
  • SPINK1 protein was confirmed to be secreted from cells in a hypoxic state and be present in and around the cells.
  • a SPINK1 Myc-tagged fusion protein expression vector or its empty vector was introduced into human cervical cancer-derived cell line HeLa and human prostate cancer-derived cell line DU145. After culturing under normal oxygen conditions for 48 hours, the cell culture fluid and the cell extract were collected and Western blotting was performed using anti-Myc tag antibody and anti-ß-Actin antibody. Forty-eight hours after the gene introduction, 0, 2, 4, or 6 Gy gamma ray irradiation was performed under normal oxygen conditions. The cells were then cultured for 14 days to perform a colony formation test.
  • FIG. 40 shows the results. As can be understood from FIG. 40 , it was confirmed that when a SPINK1 expression vector is introduced into Hela cells and DU145 cells, SPINK1 can be overexpressed in both of the Hela cells and DU145 cells. It was also confirmed that in both of Hela cells and DU145 cells, the overexpression of SPINK1 protein increases radiation resistance of the cells. Based on these results, it could be understood that SPINK1 contributes to exacerbation of a pathological condition and that SPINK1 can be an indicator of exacerbation of a hypoxia-related pathological condition.
  • Human prostate cancer-derived cell line DU145 was cultured in serum-free medium under normal oxygen (20%) conditions for 24 hours. The medium was then replaced with normal medium (containing 5% serum) containing a recombinant SPINK1 protein at a final concentration of 100 ng/ml or with normal medium not containing the recombinant SPINK1 protein. The cells were cultured under normal oxygen conditions for 24 hours. The cells were then irradiated with 0, 2, 4, or 6 Gy gamma rays and cultured for 14 days. A colony formation test was then performed.
  • FIG. 41 shows the results. As can be understood from FIG. 41 , it was confirmed that when the cells are irradiated in the presence of recombinant SPINK1 protein (rSPINK1 in FIG. 41 ), radiation resistance of the cells is induced. From these results as well, it could be understood that SPINK1 contributes to exacerbation of a pathological condition and that SPINK1 can be used as an indicator of exacerbation of a hypoxia-related pathological condition.
  • SPINK1 expression vector pCDH/SPINK1
  • pCDH-EF1-MCS-IRES-Puro SPINK1 expression vector
  • pCDH-EF1-MCS-IRES-Puro SPINK1 expression vector
  • pCDH-EF1-MCS-IRES-Puro SPINK1 expression vector
  • pCDH-EF1-MCS-IRES-Puro SPINK1 expression vector
  • pCDH-EF1-MCS-IRES-Puro pCDH-EF1-MCS-IRES-Puro
  • These viruses were infected with human prostate cancer-derived cell line DU145 and cultured in the presence of puromycin to create SPINK1 stable expressing cells (DU145/SPINK1) and negative control cells (DU145/EV).
  • mice were transplanted subcutaneously into the right lower-leg thigh of immunocompromised mice (BALB/c nu/nu) to prepare tumor-bearing mice.
  • the transplanted tumor was locally irradiated with 0 or 10 Gy gamma rays, and subsequent volume changes in the transplanted tumor were measured.
  • FIG. 42 shows the results. As can be understood from FIG. 42 , it was confirmed that in vivo as well, the radiation resistance increases in the presence of SPINK1 protein. Based on these results, it could be understood that SPINK1 contributes to the worsening of a pathological condition in a hypoxic environment.
  • Human prostate cancer-derived cell line DU145 was seeded into 96-well cell culture plates (4 ⁇ 10 2 cells per well in a 96-well plate when the dose of the subsequent irradiation is 0 Gy; and 1.2 ⁇ 10 3 cells per well when the dose is 4 Gy) and cultured in a CO 2 incubator under a normal oxygen concentration overnight. Thereafter, the cells were cultured in serum-free medium for another 24 hours and then cultured in the presence or absence of recombinant SPINK1 purified protein and in the presence or absence of anti-SPINK1 antibody under normal oxygen conditions for 24 hours. The cells were then irradiated with 0 Gy or 4 Gy gamma rays and cultured under normal oxygen conditions for an additional 3 days. Viable cells were then quantified with Cell Count Reagent SF (produced by Nacalai Tesque, Inc., Catalog #07553-44).
  • FIG. 43 shows the results. As can be understood from FIG. 43 , radiation resistance of the cells was induced in the presence of recombinant SPINK1 purified protein. When SPINK1 neutralizing antibody was allowed to act on the protein, this effect disappeared. These results confirmed that inhibiting SPINK1 can mitigate radiation resistance of cancer cells. This confirmed that inhibiting SPINK1 can inhibit exacerbation of a pathological condition caused by a hypoxic area.
  • FIG. 44 shows the results.
  • the overall survival rate is plotted on the ordinate, and the observation period (unit: year) is plotted on the abscissa, with the date on which the patient underwent tumor resection surgery being defined as 0.
  • the median value of the SPINK1 concentration of the preoperative plasma was 27.6 ⁇ g/L (range: 20.5 to 185.5 ⁇ g/L).
  • the median value of the observation period was 2.6 years (range: 1.3 to 5.0 years).
  • the obtained preoperative SPINK1 concentration data were divided into two groups, i.e., a high-value group (High in FIG.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Organic Chemistry (AREA)
  • Cell Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Veterinary Medicine (AREA)
  • Wood Science & Technology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Vascular Medicine (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
US18/574,504 2021-06-29 2022-06-20 Hypoxia biomarker and use thereof Pending US20240295567A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2021108196 2021-06-29
JP2021-108196 2021-06-29
PCT/JP2022/024612 WO2023276768A1 (ja) 2021-06-29 2022-06-20 低酸素バイオマーカー及びその利用

Publications (1)

Publication Number Publication Date
US20240295567A1 true US20240295567A1 (en) 2024-09-05

Family

ID=84691769

Family Applications (1)

Application Number Title Priority Date Filing Date
US18/574,504 Pending US20240295567A1 (en) 2021-06-29 2022-06-20 Hypoxia biomarker and use thereof

Country Status (3)

Country Link
US (1) US20240295567A1 (https=)
JP (1) JPWO2023276768A1 (https=)
WO (1) WO2023276768A1 (https=)

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006526992A (ja) * 2003-06-12 2006-11-30 ユニバーシティ オブ マニトバ 癌を検出し、癌の進行をモニタリングする方法
JP2010508855A (ja) * 2006-11-08 2010-03-25 ザ リージェンツ オブ ザ ユニバーシティ オブ ミシガン 前立腺癌マーカーとしてのｓｐｉｎｋ１およびその使用
GB0908142D0 (en) * 2009-05-12 2009-06-24 Cancer Res Inst Royal Sample cutter and production of tissue arrays
CA2817564A1 (en) * 2010-11-18 2012-05-24 Ronald K. Blackman Preselection of subjects for therapeutic treatment based on hypoxic status
JP2013545759A (ja) * 2010-11-18 2013-12-26 シンタ ファーマスーティカルズ コーポレーション 低酸素状態に基づくエレスクロモールによる治療に適した被験体の事前選択

Also Published As

Publication number Publication date
JPWO2023276768A1 (https=) 2023-01-05
WO2023276768A1 (ja) 2023-01-05

Similar Documents

Publication Publication Date Title
Gao et al. Circulating tumor cell is a common property of brain glioma and promotes the monitoring system
JP5713035B2 (ja) 癌の検出方法
Mourskaia et al. ABCC5 supports osteoclast formation and promotes breast cancer metastasis to bone
Zhai et al. Analysis of clinical factors and PDGFR-β in predicting prognosis of patients with clival chordoma
JP6281873B2 (ja) 新規癌マーカーおよびその利用
JP6271636B2 (ja) 肺癌および結腸直腸癌におけるｂａｒｄ１アイソフォームおよびその使用
Olar et al. Absence of IDH1-R132H mutation predicts rapid progression of nonenhancing diffuse glioma in older adults
Dong et al. Dexmedetomidine promotes colorectal cancer progression via Piwil2 signaling
Lin et al. NOL4L, a novel nuclear protein, promotes cell proliferation and metastasis by enhancing the PI3K/AKT pathway in ovarian cancer
US20240295567A1 (en) Hypoxia biomarker and use thereof
CN117752795B (zh) T5224在抑制肝癌侵袭、转移中的应用
WO2011129427A1 (ja) 癌の診断剤および治療剤
JP6341859B2 (ja) がんマーカーおよびその用途
KR102897399B1 (ko) 유방암 진단을 위한 신규 바이오마커
Liu et al. Deficiency in SPOP-mediated ubiquitination and degradation of TIAM1 promotes gastric cancer progression
CN111110849B (zh) 丝氨酸蛋白酶抑制因子Kazal 1型在制备细胞衰老及肿瘤诊断或调控制剂中的应用
Lin et al. Clinical significance of nucleostemin expression and its correlation with cyclin D1 expression in malignant ovarian tumors
JP7774310B2 (ja) 腎がん患者の抗がん剤抵抗性および予後予測方法、抗腎がん物質のスクリーニング方法、ならびに、腎がん治療用医薬組成物
KR102576165B1 (ko) 침윤성 뇌암 동물 모델 및 이의 제조방법
JP2008169132A (ja) Ｌｉｎ７ｃの癌の転移診断への応用
KR20230120908A (ko) 신경교종의 악성도 진단을 위한 바이오마커 및 이의 용도
WO2017186103A1 (zh) Pde3a在判断阿那格雷治疗肿瘤效果中的应用
WO2007026960A1 (ja) Ｍｏｃｓ３遺伝子の治療的又は診断的用途
US20130130931A1 (en) Biomarker for the diagnosis, prognosis and monitoring of cancer
JPWO2019156111A1 (ja) メラノーマのがん治療耐性を判定する方法

Legal Events

Date Code Title Description
AS Assignment

Owner name: KYOTO UNIVERSITY, JAPAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HARADA, HIROSHI;KOBAYASHI, MINORU;SUWA, TATSUYA;SIGNING DATES FROM 20231030 TO 20231109;REEL/FRAME:066032/0330

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION